GENE ENCODING TREfiALOSE-6-PHOSPHATE PHOSPHATASE AND USE THEREOF ,;
TECHNICAL FEELD
The present invention relates to a gene encoding trehalose-6-phosphate and use thereof in particular, a yeast for practical use with superior reastance to dryness and/or resistance property to how- storage, alcoholic beverages produced with said yeast, and a method for producing said bevK'agK. M)re particularly, the present invention relate to a yeast, whose resistance property to dryness and/or resistance property to low-temperature storage is enhancing by anrolrfying eqiression level of 'IPS2 gene encoding a proton Tps2p having a trehalose-6-phosphate phosphatase activity in brevrer's yeast, non-ScTPS2 gene specific to a lager brewing 5^ast and to a method for producing alcoholic with said yeast, etc. Further, the yeast of the present inrention is useful as a baker's yeast or an industrial yeast as well.
BACKGROUND ART

"Renjo" may be terminated at a certain times of fermentation is carried out The number of times of "Renjo" may vary according to fermaitation conditions or properties of yeasts used m the process. A process to develop yeasts for fermentation is called propagation. YeaSs are
Concerrnng a method for producing dry yeast maintaining high viable cell ratio, improvement of drying or improvement of manicuring conditions such as temperature or addition of emulsifiers, etc., have been made. For example, L-drying method is not

practical to be used at industrial production scale because, though it can maintain esrremely high ™ble cell ratio, biJt at tiie same lime it takes a lot of time and cost.
Regarding low-temperature resistance of yeast, some experiments designed to improve re&igeration-r^stant property mainly of baker's yeast were reported. This is because Saccharomyces cerevisiae, which is a baker's yeast, has poor low-temperature storage property in comparison with brewesr's yeast for bes or sake, which can ferment at low temparature. For fficample, bakra-'s yeasts having refiigeration-resistant property and drying-resistant property were fouwi out mainly by screeoii^ methods in Japanese PateittAj^lication Laid-open No. HU-155559 and Japanese Patent Application Laid-open No. 2003-304864. Furtha, regarding exan^Ies utilizing gKiaic eo^neaii^ techniques, trehalose hi^y accumulating strains by disruption of NTHl, which is a trehalase gene, is reported in Japanese Patent Apphcation Laid-open No. H10-117T71 and a strain highly accumulatii^ specific amino adds such as arginine by disruption of CARl, which is an argjnase gene, is reported in J^anese Patent Application Laid'Kjpen No. 2001-23 S665.
DISCLOSURE OF INVENTION
Under the above atuations, there has been a need to make hi^efBciency production of alcoholic beverages or useful materials possible by using a gene encoding a protein responsible for drying and/or low-temperature storage-resistant property bf brewery yeast and said protein.
The present inventors made extensive studies to solve the above problems and as a result, succeeded in identiiying and isolating a gene aicoding trehalose-6-phosphate phosphatase from beer yeast. Moreover, the present inventors produced transformed yeast in which the obtained gene was fflqjressed to vaify that drying-reastarrt property and/or low-temperature storage^eastant property can be acQially improved, therdjy completing the present invention.
Thus, the present invention relates to a gene Kicoding a trehalose-6-phosphaie phosphatase of brewery yeast, to a protein encoded by said gene, to a transfonned yeast in which the ffiq)ression of said gene is controlled, to a method for enhancing drying-resistant prqserty and/or low-temperature stOTage-resistant property of yeast tising a yeast in w^ch tiie expression of said gene is controlled, or the like. More specifically, the present invention provides the following polynucleotides, a vector con^rising said polynucleotide, a transformed yeast introduced witii said vector, a method for produdng alcoholic beverages by using said transformed yeast, and the like.
(1) A pofynucleotide selected from the group consisting of
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDNO:!;

(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(c) a polynudectide comprising a polynucleotide encoding a protem consisting of the amino add sequence of SEQ E) NO: 2 in vfinch one or more amino adds th^eof are ddeted, substituted, inserted and/or ad^d, and having a trehalose-6-phosphate dephosphoiyiation activity,
(d) a polynucleotide comprising a polynucleotide encoding a protan having an amino add sapience having 60% or higher identity witii tiw ammo add sequraice of SEQ ID NO: 2, and said protdn having a trdBlose-6-phosphate dq)hoq)hoiylation activity;
(e) a polynucleotide comprising a polynucleotide wiich hybridizes to a pofynucleotide consisting of a nudeotide sequence complementary to the nucleotide sequaice of SEQ ID NO: 1 under &imgeat conditions, and wtiich encodes a protdn having a trehalo^-6-phaspbate dephosphoiyiation activity, and
(f) a polynucleotide conrprising a polynucleotide \^ch i^jbrifces to a pdyaucleotide consisting of a nudeotide sequKice complementary to the nucleotide sequence of the polynucleotide encoding the protdn havir^ the amino acid sequence of SEQ ID NO: 2 under stringent conditions, and which encodes a protdn having a trdialose-6-pk)sphate dephosphoiyiation activity.
(2) The polynudeotide according to (1) above selected from die group consisting of:
(g) a polynucleotide con^rising a polynucleotide «icoding a protdn consisting of the
amino add sapience of SEQ ID NO: 2, or encodii^ thd amino add sequence of SEQ ID NO: 2 in
which 1 to 10 amino adds thereof are deleted, substituted, inserted, juid/or added, and wherein said
protein has a trehalose-6-phosphate dephosphoi)1ation activity;
(h) a polynudeotide comprising a polynucleotide encodmg a protein having 90?^ or h^er identity with the amino add sequaice of SEQ ID NO: 2, and havmg a trehalose-6-phosphate dephosphoiyiation activity, and
(i) a polynudeotide comprising a polynucleotide vAmh hybridizes to a polynucleotide consisting of a nudeotide sequence of SEQ ID NO: 1 or which hybridizes to a polynucleotide consisting of a nudeotide sequence complementary to the nudeotide sequence of SEQ ID NO: 1, under high stringent conditions, vi*ich encodes a protdn havmg a trd)alose-6^ho^hate dephosphorylation activity.
(3) The polynucleotide according to (1) above comprising a polynucleotide consi^ing of the nudeotide sajuence of SEQ ID NO: I.
(4) The polynucleotide according to (1) above conqmsing a polynudeotide encodmg a protein consisting of the amino add sequence of SEQ ID NO: 2.
(5) The polynucleotide according to any one of (1) to (4) above, wherein the
polynucleotide is DNA.

(6) A protem encoded by the polynucleotide according to any one of (1) to (5) above.
(7) A vector containing the polynucleotide according to any one of (1) to (5) above, (7a) The vector of (7) above^ which comprises the expression cass^te comprising th
foOowing components:
(x) a promoto" that can be transcribed in a yeast cell;
(y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense or antisense direction, and
(z) a signal that can fimction in a yeast with respect to transcription termination and polyadenyiation of a KNA molecule.
(7b) The vector of (7) above, vdiich comprises the ejqiression c^sette comprising the following coirqronents:
(x) a promoter that can be transcribed in a yeast cell;
(y) any of the polynucleotides described in (1) to (5) above linked to the promote in a sense direction; and
(z) a signal that can fimction m a yeast with respect to transcription termination and polyadenyiation of a RNA molecule.
(8) A yeast into w^ch the veaor according to ai^ one of (7) to (7b) above has been introduced.
(9) The yeast (yeast for practical use) according to (S) above, ^^ein drying-resistant property is increased. The "yeast for practical use" means that a yeast v^^ich possesses praoical value such as brewer's (breway) yeast, baker's yeast or industrial ye^ etc,
(10) The yeast according to (8) above, wherein low-ten^seratare storage-reastant
propsty is increased,
(11) The yeast accordii^ to (9) above, whoein the dryii^-resistant property is increased by increasing an e^iression level of the protein of (6) above.
(12) The s^ast accoK^ng to (10) above, wheran the low-tHiqjerature storage-resistant property is increased by increaang an expression level of the protein of (6) above.
(12a) The yeast according to any one of (9) to (12) above, wherein the yeast is a brewery yeast.
(13) A method for producmg an alcoholic beverage by using the yeast according to any one of (8) to (12a) above.
(14) The method according to (13) abov^ wheran the brewed alcoholic beverage is a malt beverage.
(15) The method M:cording to (13) above, who-an the brewed alcoholic beverage is wine.

(16) An alcoholic be\^rage produced by the m^hod according to any one of (13) to (15) above.
(17) A method for assesang a t^ yeast for its drying-resistant property and/or low-temperature stora^resistant property, comprising usii^ a prin^r or probe designed based on the nucleotide sequence of a gene having tlM nudeotide sequaice of SEQ ID NO: I and encodirg a trehalose^pho^hate phosphatase,
(I7a) A mediod fer selecting a yeast having an increased diying-resistant prcgserty and/or low-tempa'ature storage-resistant propeaty by using the method described in (17) above. ■
(17b) A method for producing an alcoholic beverage (for example, beer or alcohol for indiisttial use, eSc.) by usii^ lite yeast aekcted vrith 1iie method de^bed i^
(17c) A method for produdng an usefiil materials (forexample, protein) by using the yeast selected with the method described in (17a) above.
(18) A method &x assesang a test yeast for its drying-resistant propKty and/or
low-temperature stoi^e-resistarit iffoj^rty, comprising: culturing the test yeast; and measuring the
expresaon level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a
trehalose'6-phosphate phosphatase.
(18a) A method for selecting a yeast having a high drying-reastant propaty and/or low-teraperatuie storage-resistant iffopeity, which comprise assessing a t^ yeast by the method described in (18) above and sdecting a yeast having a high ©ipression level of gene encoding a trdialose-6-phosphate phosphatase.
(18b) A method fia: producing an alcoholic beverage (for example, beer) by usii^ the yeast sdected with the meShod desoibed in (18a) above.
(18c) A method for producing an us^ material (for example, protran) by using the yeast selected with the mrthod described in (18a) above.
(19) A mediod fra- sdecting a yeast, comprising: culturing test yeasts; quantifying the pro^n of (6) abcfve or measuring the expresaon level of the geiw having the nucleotide sequence of SEQ ID NO: 1 and encoding a irehalose-6-phosphate phosphatase and selecting a test yeast having an amount of ihe piai^ cff the gene expression level according to &vorable drying-resistant propaty and/or low-tonperature stora^reastant property.
(20) The method for selecting a yeast according to (19) afaow, con^sing; culturing a reference yeast and test yeasts; measuring &x each yeast the ejqn^ssion level of the gene havii^ Has nudeotide sequence of SEQ ID NO: 1 and encoding a trehalose-6^ho^hate pho^hatase; and sdecting a test yeast having tiie gene e^jresaon higher than that in the reference yeast
(21) The method for selecting a yeast according to (19) above, comprising: cuhuring a reference yeast and test yeasts; (juantifying the protean according to (6) above in each yeast; and

selecting a test yeast kmng a largK" amount of the protein than that in the referaice ye^.
(22) A method for producing an alcoholic beverage comprismg: conducting femaentation usmg the yeast according to any one of (8) to (12a) above or a yeast selected by the methods according to ary one of (19) to (21) above.
The transformed yeast of the present invention is able to keep hi^ viable cell ratio during dry storage or low-temperature storage. Therefore, wdien it is used for brewing and so on, painfiilness of conserving yeast can be eliminated. Further, it is expected to contribute to quality stabilization Nforeo\«r, dry yeast is suitable for long-storage, and it is very advantageous to distribution or transportation due to its reduced weight. It is also useful as microorganisms for industrial application such as industrial alcohol production or production of useful proteins. The yeast of the present invention also useful as an industrial yeast as wsH.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the cell growth with time upon beer fermentation test The horizontal axis represents fermentation time while the vertical axis rqjresents optical density at 660 nm (OD660).
Figure 2 shows the extract (sugar) consumption with time upon beer fermerSation test
The horizontal axis represents fermentation time while the vertical axis represents ^parent extract
concentration (w/w%). '
Figure 3 shows the ffitpression profile of non-ScTPS2 gene in yeasts upon beer fermentation test. The horizontal aas represents fermentation time while the vertical axis represents the fartena^ of detected signal.
Figure 4 shows the result of drying-resistant propoty test of parent strain and non-ScTPS2 highly expressed strain.
BEST MODES FOR CARRYING OUT THE INVENTION
The present inventors isolated and identified non-ScTPS2 gene encoding a trdialose-6-phosphate phosphatase of brewery yeast based on the lager brewing yeast genome information tn^jped according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-283169. The nucleotide sequaice of the gene is represented by SEQ ID NO; 1. Furtiia-, an amino acid sequaice of a protein encoded by the gene is represented by SEQ ID NO: 2.
1. Polynucleotide of the inventJon
First of all, the present invention provides (a) a polynucleotide comprising a polynucleotide ofthe nucleotide seqtKnce of SEQ ID NO: 1; and,(b) a polynucleotide comprising a polynucleotide

encoding a protein of tiie amino add sequence of SEQ ID NO: 2. The polynucleotide can be DNA orRNA.
The target polynucleotide of the present invention is not limited to the polynucleotide encoding a protein having a trehalose-6-phoq)hate d^hosphoiylation activity described above and may include other polynucleotides ^icoding protdns having equivalent functions to said protdn. Protons whh equivalent functions include, for example, (c) a protran of an amino add sequoice of SEQ ID NO: 2 with one or more amino adds thereof bdi^ ddeted, substituted, insaled and/cr added and having a trebalDse-6-phosphate dephosphoiylation adivity.
Such proteins mchide a protein conasting of an amino add sequence of SEQ ID NO: 2 witii, for example, 1 to 100,1 to 90, 1 to 80,1 to 70,1 to 60,1 to 50,1 to 40, 1 to 39,1 to 38, lto37, lto36,lto35,1 to34,1 to 33,1 to 32,1 to 31,1 to30,1 to29, 1 to28,1 to27,1 to26,1 to 25,1 to 24,1 to 23,1 to 22,1 to 21,1 to 20, 1 to 19,1 to 18,1 to 17,1 to 16,1 to 15,1 to 14, 1 to 13,1 to 12, 1 to 11,1 to 10,1 to 9, 1 to 8,1 to 7,1 to 6 (1 to several amino ^ds), 1 to 5, 1 to 4, 1 to 3,1 to 2, or I amino add residues thereof b^g dd^d, sub^ituted, inserted and/or added and having a trebalose-6-phosphate dephosphcrylation activi^. In general, the number of ddetions, substitutions, insertions, and/or additions is p'e&rably smalls. In addition, ^ch [nxitdns include (d) a protdn having an amino add sequence with about 60% or higher, about 70% or higher. 71% or higjier, 72% or h^her, 73% or higher, 74% or higher, 75% or hi^er, 76% or higher, 77% or higher, 78% or higher, 79% or hi^er, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% OT H^ier, 85% or higher, 86% or higher, 87% or higher, 88% or Mgha:, 89% or hi^er, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96'yo or higher, 97% or higher, 98% or hi^er, 99% or higha:, 99.1% or highw, ^.2% or higher, 99.3% or higher, 99.4% or b^ha:, 99.5% w hi^ier, 99.6% or higher, 99.7% or higher, 99.8% or h^er, or 99.^/o or higher identity with the amino add sequence of SEQ ID NO: 2, and having a trehalose-6-phosphate dephos5>hoiylation activity. In general, the percentage identity is preferably highK".
Trehalose-fr^hosphate phosphatase activity may be measured, for exan^ile, by a method described in Eur J Biochem 1993 Mar 1; 212(2): 315-23.
Furthermorei, the presait inveittion also contanplats (e) a polynucleotide comprismg a polynudeotide vMch hybridizes to a polynudeotide conasting of a nudeotide sequence con^jlementaty to the nudeotide sequence of SEQ ID NO: 1 under stringent ronditions and vrfikh encodes a inotdn having a trebabse-6-phospbate dephosphoiylation actiyity, and (f) a polynudeotide compiisii^ a polynucleotide vMcb hybridizes to a polynudeotide complementaiy to a nudeotide sequence of encoding a protdn of SEQ ID NO: 2 under stiingart conditions, and i«diich encodes a prat&n having a trebalose-6-phosphate depbosphoryktion activity.
Herdn, "a polynudeotide that hybridizes under sringent conditions" refers to nudeotide

sequence, aich as a DNA, obtained by a colony hybridization technique, a plaque hybridization technique, a southon hybridization technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd Ed., CURRENT PROTOCOLS m MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997, and so on.
The tmm "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardes solution, 0.5% SDS, 50% fonnamide at 32°C. "Moderate stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formaniide at 42''C. "H^ stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50X. Under these conditions, a polynucleotide, such as a DNA, with higher homology is e5q)ected to be obtained efficiently at higher temperature, although multiple fectOTs are involved in hybridization stringency includmg temperature, probe concentration, probe length, ionic length, time, salt concentratiai and othea^, and one skilled in the art may appropriately select these Actors to realize similar stringency.
When a commwciaEy available kit is used for l^bridization, for example, Alkphos Direct Labelir^ Reagents (Amersham Pharmacia) may be used. In this case, accordmg to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (wA^) SDS at 5 5°C, tha-eby detecting hybridized polynucleotide, such as DNA.
Other polynucleotides that can be hybridized include polynucleotides having about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or bi^er, 75% or higher, 76% or lu^er, 77% or higher, 78% or higher, 79% or higher, 80% or higher, S1% or highra", 82% or higher, 83% or Hgher, S4% or higha", S5% or higher, 86% or hi^er, 87% or higher, 88% or hitler, 89% or higher, 90% or higher, 91% or hi^ier, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or hi^er or 99.9% or higher identic to polynucleotide encoding the amino add sequence of SEQ ID NO: 2 as calculated by homology search software, such as FASTA and BLAST using defeuh parameters.
Identity between amino add sequences or nudeotide sequences may be determined using algoritiim BLAST by Kariin and Altschul (Proc. Natl Acad Sci. USA, 87: 2264-2268, 1990; Proc. NalAcadSd. 054,90:5873,1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et al., J. Mol Biol 215: 403, 1990). When a

nucleotide sequence is sequenced using BLASTN, the parameters are, ftff" example, score = 100 and v/oid length = 12. When an ammo ^d sequence is sequenced using BLASTX, the parameto^ ar^ for example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, defeuh parametKS for each of the programs are employed.
2. Protein of the oresentiiivieirtion
The presart invention also provides proteins encoded by any of the polynucleotides (a) to (i) above. A preferred protein of tiie present invaition conqjrisK an amino acid sequence of SEQ ID NO; 2 with one Cff several amino acids thereof being detettd, substituted, inserted and/or added, aud having a trdialose-6-pbosphate d^hosphorylation actrtoty.
Such protdn includes those having an amino add sequence of SEQ ID NO: 2 with amino add residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a trehaIose-6-phospha£e dephosphor>dalion activity. In addition, such protein indudes those having homology as desoflaed above with the amino add sequence of SEQ ID NO: 2 and having a trehaIose-6-phosphate dephosphorylalion activity.
Such protons may be ohained by emplojdng site-directed mutation desmbed, for Ktanqjle,. in MOLECULAR CLCMING 3rd Ed, CURRENTPROTOCOLS INMOLBCULARBIOLOGY, Niic. Acids. Res., 10: 6487 (1982), Proc. Natl Acad Sci. USA 19: 6409 (1982), Gem 34:315 (1985), Afe. Acids. Res., 13:4431 (1985),Prcc. Natl Acad Set USA 82:488 (1985).
Deletion, substitution, insertion and/or addition of one or more amino add readues in an amino add sequoice of the protein of the invention meai^ that one or more amino add residues are ddeted, substituted, inserted and/or added at any one or more podtions in the same amino add sequfflice. Two or more types of ddetion, substitution, insertion and/or addition may occur concurrently.
Heranafta-, exangjle of mutually substitutable amino add residues are exHunerated Amino add residues in the same groiq) are mutually substitutable. The groups are provided below
Group A: leucine, isoleudne, norieudne, valine, norvaline, alanine, 2-aminobutanoic add, methionine, o-methylsraine, t-butj4glycine, t-bu^lalanJne, cycldhej^^Ialanine; Group B: asparatic add, ^utamic add, isoasparatic add, iso^utamic add, 2-aiiiinoadipic add, 2-amino8ub^c add; Gnaup C: asf)ar^jne, j^utamine; Group D: lysine, argnine, ornithine, 2,4-diaiDinobutanoic add, 2,3-diaminDpropionic add; Group E: i^oline, 3-hydrcBq'prDline, 4-hydro;q^roline; Group F: serine, threonine, homosaine; and Group G: phenjrtalanme, tyroane.
The protdn of the present invention may also be px)duced by chemical syntiiesis methods such as Fmoc method (fluorenyhnethjdffiQ'rarbonyl method) and ffioc method (t-butyloxycari)or^l methoi^. hi addition, pe^Jtide syntiiesizers amiable from, for exanqjle. Advanced ChemTedi,

PerkinElmer, Pharmacia, Protan Technology Listrument, Synthecell-Vega, Pa-Septive, Shimaai Ccap. can also be used for chemical synthesis.
3. Vector of the invention and veast transfonned with fee vector
The present invention then provides a vector comprising the polynucleotide describL.^ above. The vector of the prraent invention is directed to a vector mduding any cf the polynucleotides described in (a) to (i) above or any of the polynucleotides described in (j) to (m) above. Generally, the vector of the presait invention comprises an expression cassette including as con^onents (x) a promoter that can transcribe in a yeast cell; (y) a polynucleotide described in any of (a) to (i) above that is linked to the promoter in sense or antisense direction; and (z) a signal that fimctions in the yeast with respect to transcription termination and polyadenylation of RNA molecule. Further, in order to highly express the protdn of the invention, these polymicleotides are preferably introduced in the sense direction to the promots" to promote e^resaon of the polynucleotide (BNA) desaibed in any of (a) to (i) above.
A \^aor introduced in the yeast may be any of a multicopy type (YBp type), a single copy type (YCp type), or a chromosome integration type (Yip type). For example, YEp24 (J, R. Broach et al,, EXPEEaMENTALMAMPULATlONOFGENEEXPRESSlCM, Academic Press, New York, 83, 1983) is known as a YEp type veaor, YCpSO Qsi. D. Rose et al.. Gene 60; 237, 1987) is known as a YCp type vector, and YIpS (K. Stmhl et al,, Proc. Nail Acad Sci USA, 76: 1035, 1979) is known as a Yip type vector, all of whidi are readily available.
Promotere/temiinators fOT adjustii^ gene expression in yeast may be in ai:^' combination as bng as thefy function in the yeast for practical use and they are not influenced by constituents in fermentation broth. For exanqjle, a promoter of glyceraldel^'d^ 3-phosphBfle dehydrogenase gene (TDH3), OT a promoter of 3-phosphogiycerate kinase gene (PGKl) may be used. These genes have previously been cloned, desa&ed in detail, for example, m M. F. Tuite et al., EMBO J., 1, 603 (1982), and are readily avmlabie by known metkjds.
Since an auxotropl^ raaricer cannot be used as a selective marker upon transformation for a yeast for practical use, fer example, a genetidn-resistant gene (G418r), a coppa'-resistant gene (CUPl) (Marin et al., Proc. Natl Acad Sci. USA, 81,337 1984) or a cerulaiin-resistant gene (fas2m, PDR4) (Junji Inokodu et al., 5jDc/Kmi5irv, 64, 660,1992; and Hussain et al.. Gene, 101:149,1991, respKtrvely) may be used.
A vector constructed as described above is introduced into a host yeast. Examples of the host yeast include ai^ yeast ^^east for practical use) that can be used for brewing, for example, brewery yeasts for beer, wine and sake, baker's yeast, ye^ for producing industrial alcohol or yeast for produdng use&l proteins and so on Spedfically, yeasts such as genus Sacdiaromyces may be

used. According to the present invention, a iager brewing yeast, for example, Saccharomyces pastcrianus W34/70, etc., Saccharontyces carhbergensis NCyC453 or NCYC456, etc., or Saccharomyces cerevisiae 'NB'RC1951, >JBRC1952, NBRC1953 or NBRC1954, etc., may be used. In addition, ift^iisl^ yeasts such as Sacchvomyces cerevisiae NCYC90, wine yeasts such as wine yeasts #1,3 and 4 ftcan the Brewing Sodety of Japan, and sake yeasts sudias sakeyrast#7 and 9 from the Brewing Sodety of Japan, baker's yeast such as NBRC0555,3*JBR.C1346 or NBRC2043, etc., may also be used but not limited ther^. In the present invention, lager brewing yeasts such as Saccharomycespastarianus may be used preferably.
A yeast transformation method may be a generally used known method. For example, methods that can be used include but not limited to an electroporation method Q^e^ Emym., 194: 182 (1990)), a spheroplast method {Proc. Nail Acad Sci USA, 75: 1929(1978)X a Bthium acetate mdhod (J. Bacteriology, 153:163 (1983)), and methods described m/"roc. A'o^Z^caiijS'ci USA, 75: 1929 (1978X METHCDS IN YEAST (^^Encs, 2000 Edition: A Cold Spring Harbor Laboratcay Course Manual
More qjedfically, a host yeast is cultured in a standard yeast nutrition medium (e.g., YEPD medium (Gsietic Engineering. Vol. 1, Plenum Press, New Yoric, 117(1979)), etc.) aidi that OD600 nm will be 1 to 6. This culture yeast is collected by ceatrifugation, washed and pre-treated with alkali metal ion, preferably lithium ion at a concentration of about 1 to 2 M After the cell is left to stand at about 30°C for about 60 minutes, it is left to stand withDNA to be introduced (about 1 to 20 ^g) at about 30°C for about anotha- 60 minutes. Pofyethylen^ycol, preferably about 4,000 Dahon of polyetbylen^lycol, is added to a final concentration of about 20% to 50%. After leaving at about 30°C for about 30 mmutes, tiie cell is heated at about 42°C for about 5 minutes. Preferably, tins cell suspension is wadied witii a standard >«ast nutrition medium, added to a predetermined amount of fresh standard yeast mitrition medium and left to stand at about 30°C for about 60 minutes. Thsres^sr, it is seeded to a standard agar medium containing an antibiotic or the like as a selective maifcer to obtain a transfonnant
Other general cloning techniques may be found, for example, m MOIBXILAE dCiHING 3rd Ed., andMETHCOSlNYEAOTC^sIElKS, A LABCJRATORY MANUAL (Cdd Spring Harbor LaboratMy Press, Cold Spring Harbor, NY).
4. Method of producing alcoholic beveragK according to the present invention and alpoholic beverages nrodaced bv the method
A yeast having a supaior dijdng-reastant property and/or low4emperature stcff^e-i^stant propearty can be obtained by introdudng tiie vector of the present invention described above to a yeast. Further, a yeast having a si^erkff diying-resistant property and/or

low-temperature storage-resistant property can be obtained by selecting a yeast by the yeast assessment method of the present invention described below. The target use of yeasts obtained in the present invention include, for example, but not limited to, brewing alcoholic beverages such as beer, wine, whisky, sake and the like, baking bread, manufecturii^ usefiil materials such as industrial alcohol production and production of use&l proteins.
In ordCT to produce these products, a known technique can be used except that a yeast fq pr^cal use obtained according to the present invention is used in the place of a parent strain. Since starting mataials, manufecturing equipment, manufecturing control and the like may be the same as the conventional ones; it can be perfiiimed without increasmg cost.
5. Yeast assessment mefliod of the invoition
The present invention relates to a method for assessing a test yeast for its drying-resistant property and/or low-temperature storage-resistant piopaty by usmg a prima- or a probe designed based on a nucleotide sequence of a gene having the nucleotide sequence of SEQ E) NO: 1 and encodii^ a trehaIose-6-pho^hate phosphatase. Gaieral technique for such assessmait method is known and is described in, for example, WOOl/040514, Japanese Laid-Open Patent Application No. H8-205900 or tlK Uke. This assessment method is described m below.
First, genome of a test yeast is prepared. For this preparation, any known method such as Hereford method or potasdum acetate method may be'used (eg., METHCDS m YEAETT GENEHCS, Cold Spring Ifeihor LaboiBtory Press, 130 (1990)). Usmg a primer or a probe designed based on a nucleotide sequence (preferably, ORF sequence) of the gene encoding a trehalose-6-phosphate phosphatase, the existence of the gene or a sequence spedfic to the gene is determined in the test yeast genome obt^ed. The primer or the probe may be designed according to a known technique.
Detection of the gene or the spedfic sequence may be carried out by employing a known technique. For exan:q>le, a polymicleotide including part or ail of the specific sequence or a polynucleotide mcluding a nudeotide sequence con:^)lementary to said nucleotide sequence is used as one primer, vM.s a polynucleotide including part or all of the sequmce upstream or downstream from this sequKice or a polynucleotide including a nucleotide sequence complementary to said nucleotide sequence, is used as anotiia- primer to amphfy a nucleic add of the yeast by a PCR method, tha-djy determining the existence of an^lified products and molecular wdght of the amplified products. The number of bases of polynucleotide used for a primer is generally 10 base pairs (bp) or more, and preferably 15 to 25 bp. hi general, the number of bases between the primers is suitably 300 to 2000 bp.
The reaction conditions for PCR are not particularly limited but may be, for example, a denaturation temper^ure of 90 to 95°C, an annealmg temperature of 40 to 60°C, an elongation

temperature of 60 to 75°C, and the number of cyde of 10 or more. The resulting reaction product may be separated, for ©ample, by electrophoresis using agarose gel to detamine the molecular wei^t of the ampUfied product. This method allows predicticm and assessment of the diyjng-resistant property and/or low-temperature storage-resistant prop^ty of yeast as determined by whetha- the molecular weigjit of the amplified product is a size that contains the DNA molecule of the sped5c part. In addittoo, by analyzing die nucleotide sequence of the amplified product; the propaty maybe predicted and/or assessed mta-e precisely.
MoreovH", in the i»esait invention, a test yeast is cultured to measure an expression level of the gaie encodit^ a trdialose-6-phosphate phosphatase having the nudeotide sequence of SEQ ID NO: 1 to assess the test yeast for its drying-resistant propa:ty and/or tow-temperature stOTage-resistam property. Measuranent of e^rrasion level of the gene encoding a trehalose-6-phosphatase can be pafonned by culturing test yeast and then quantifying mRNA or a protan resulting ftom the geiK. The quaiflification of mRNA or protean may be carried out by employing a known technique. For example, mRNA may be quantified, by Northern l^bridization or quantitative RT-PCR, while protem may be quantified, for exan^Ie, by Western blotting (ClTREH^ PROTOCOLS INKfomcUDUl BIOLOGY, John Wiley & Sons 1994^
FurthomME, test yeasts are cuhural and ej^ffession levels of the gene encoding a trehalose-6-phosphate phosphatase having the nucleotide sequaice of SEQ ID NO: 1 are measured to select a test yeast with the gene e^resaon level according to the target trehalose-produdng ability, therdjy a yeast fevorable for brewing desired alcohdic beverages can be selectal. In addition, a reference yeast and a test yeast may be cultured so as to measure and compare the ejqffession level of the gene in each of the yeasts, thaeby a fevorable test yeast can be sdected. MDTC giecifically, for example, a reference yeast and one or more test yeasts are cultured and an ^iression level of the gene encoding a trdialose-6-phosphate phosphatase havmg the nudeotide sequence of SEQ ID NO: lis measured in eadiyea^ By sdecting a tea yeast with the gaie expressed hi^a-than thai in the reference yeast, a yeast suitable for brewing deared alcoholic beverages or production of usdiil materials can be selected.
Altemativdy, test yeasts are cultured and a yeast with a high trehalose-producang abihty is selected, thffdsy a yraS suitable for brewmg desired alcoholic beverage or production of usefiil mateiiais can be selected.
In tiiese cases, the test yeasts or the refa-ence yeast may be, for example, a yeast introduced with the vector of the invention, an artificially mutated yeast or a naturally mutated yeast The trehalose-6-phosphate phosphatase activhy can be measured by, for example, a method destaibed in Eur J Biochem. 1993 Mar 1: 212(2): 315-23. The mutation treatment may employ any metiiods mcluding, for example, physical methods such as uhmviolet irradiation and radiation irradiation, and

chemical methods associated with treatments with drugs such as EMS (ethyhnethane sulphonate) and N-methyl-N-nitrosoguanidine {see, e.g., Yasuji Oshiina Ed., BIOCHEMISTRY EXPERIMENTS vol, 39, YeastMoleculai-GeneticExperimenls^i^. 67-75, JSSP).
In addition, examples of yeasts used as the reference yeast or the test yeasts include any yeasts (yeasts for practical use), for example, brewery yeasts for beer, wine, sake and the like or baker's yeast, yeast for producing industrial alcohol or yeast for producing useful protdns, etc. More specifically, yeasts such as genus Saccharomyces may be used (e.g., S. pastonarms, S. cereviMae. and S. carbbergenMs). According to the present in-rontion, a lager brewing j^ast, for example, Sacdiaromyces pasioiimivs W34/70; Sacchiromyces carlsbergensis NCYC453 or NCyC456; or Saccharoniyces cerevisiae NBRC1951, NERC1952, NBRC1953 orNBRC1954, etc., may be used. Further, wine j^asts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japan; and sake yeasts such as sake yeast #7 and 9 from the Brewing Sodety of Japan, baker's yeast such as NBRC055S, NBRC1346 and NBRC2043, etc., may also be used but not Umited tho-eto. In the present invention, lager brewii^ yeasts such as Saccfiaromyces pastoriatms may preferably be used. The reference yeast and the test yeasts may be selected from the above yeasts in any combination.
EXAMPLES
Hereinafter, the present invention will be described in more detail with reference to working examples. The present invention, however, is not limited to the examples described below.
F,TamDle 1: Onnitif^ nf Gene Encoding Trehalose-6-phosphate phosphatase (non-ScTPS2>
A gene encoding a trehalose-6-phosphate phosphatase of lager brewing yeast (non-ScTPS2) (SEQ ID NO: 1) was found as a result of a search utilizing the comparison database described in Japan^e PataE Application Laid-open No. 2004-283169. Based on the a:quired nucleotide sequence information, primers non-ScTPS2_fir (SEQ ID NO: 3) and non-ScTPS2_rv (SEQ ID NO: 4) wa-e deagned to amplify the foil-length of llie gene. PCR was carried out uai^ chromosomal DNA of a genrane sequencing strain, Sacchaiomycra pastorianus Weihenstephan 34/70 (sometimes abbreviated as "W34/70 strain"), as a template to obtain DNA fragments including thefiill-Iength gene of non-ScTPS2.
The non-ScTPS2 gene fegments thos obtained wea^ hiserted into pCR2.1-T0P0 vector (Ihvitrogen) by TA clonu^. The nucleotide sequences of the non-ScTPS2 gene were analyzed by Sanger's method (F. Sanger, Science, 214:1215,1981) to confirm the nucleotide sequence.

Example 2: Analysis ctfExpressinn of nnn-ScTPSl Gene during Beer Jermentatioo
A beer fermentation test was conducted using a lager brewing yeast, Saccharomyces pastorianus M^4/70, and niRNA extracted from the lager brewing yeast during fermentation was detected by a beer yeast DNA microair^.
Wort extract concentration 12.69%
Wortcontait 70 L
Wort dissolved osygen concentration 8.6 ppm
Fermentation temperature 15°C
Yeast pitching rate 12.8x10^ cells/mL
The fermentation liquor was san^)Ied over time, and tie time-course dianges in amount of yeast cdl growth (Fig. 1) and appareat extract concentration (Fig. 2) ware observed. Simultaneously, yeast cells wse sampled to prepare mRNA, and the pr^ared mRNA was labeled with biotin and was hybridized to a beer yeast DNA microarray. The signal was detected using GraieChip Opmiting system (GCOS; GeneChip Operating Software 1.0, manu&ctured by A^metrix Co). ]&qjression pattan of the non-ScTPS2 gene is shown in F^ure 3. This resait confirmed the ejqresision of the non-SeTPS2 gene in the general bee- feraentation.
Eiample 3; Construction of noD-ScTPS2 Hiehlv Expressed Strain
The non-ScTPS2/pCR2.1-TOPO described m Sample 1 was digested with the restriction aizymes SacI and NotI to jffqMie a DNA fragment containing the aitire length of the proteiii-eiK»ding region This fegmait was ligated to pYCGPYNot treated with &e restriction raizymes Sad and Noli, iher^ constructing the non-ScTPS2 high expression vector Don-ScTPS2/pyCCH'YNca. pYCGPYNot is a YCp-type yeast ejqjression vector. A gsas insMted is highly expressed by the pynivate Idnase gene PYKl promoter. The graietidn-resistant gene 0418^ is included as the sdectable maricer in the yeast, and the an:q)icillin-resistant gene Amp' as the selectable marker m Esdieiidiia coli.
Using tlte hi^ ejqjresaon vector prqiared by the above inethod, an AJL4004 strain was transformed by the method described in Japanese Patent Application Lad-open No. H07-303475. The transformants were selected on a YPD plate medium (1% yeast extract, 2% polypqitone, 2% gjucose and 2% agar) containing 300 mg/L of geneticin.
Example 4: Evaluation of I)rvinfi.rwii!rftmt Propalv of nQn-ScTPS2 Hif^hly Ryprpgapii Strain

Drying-resistant properties of the parent strain (AJL40O4 ^rain) and the non-ScTPS2 highly expressed strain obtained by the method described in Example 3 were evaluated by a method described below.
One platinum loopfiil of each yeast was inoculated into 10 mL of wort containing 100 xng/L of geneticin, and stirred at SCC overnight (precultivation). The precultivation liquid was inoculated into 10 mL wort containing 100 mg/L of geneticin to make its OD660 = 0.5, thai main culture was initiated. The culture was continued for 2 days until the growth of the yeast reached stationary phase. Turbidity of the culture was measured at the completion of the culture, then the culture liquid was suspended in sterile water to make its OD = 2. One hundred microliter (100 |jL) of the suspension thus obtained was dispensed into a 1.5 mL microtube, then the yeast cells were dried by evaporation for I hour using a reduced-pressure' concentrator (DNAllO SpeedV^ (re^sto'ed trademark), manufactured by ThennoSavant).
Viable cell ratio was measured by a method described below. The dried yeast cdk obtained above -wsre resu^ended in 50 [JL of sterile water, then 50 (JL of 0.02% roethjdene blue solution (pH 4.5) was added to the susprasioa Blue-stained yeast cells which had lost reducing powa" were considered as dead yeast cells. Then the suspaision was observed under a microscope, and viable cell ratio was measured usmg a Cell Vital Analyze" Sj^stem (DA cdl counta:, manu&ctured by Yamato Scientific Co., Ltd.). The cells were counted until the population reached more than 2000 cdls to minimize experimental error. '
As indicated in Fi^ire 4, viable cell ratio of the highly-expressed strain vras 35.9%, though viable cell ratio of the parent strain was 19.9%. It w^ demonstrated by the results that dr^g-resistant propaty of yeast was increased by high expresdon of non-ScTPS2.
Example 5: Evaluation of Low-temperature Resistant Property of DQn-ScTPS2 Hifrhlv Expressed Strain
Low--^nperature rraisiant property of the parent strain (A3L4004 strMi) and the non-ScTPS2 highly expressed strain obtained by the method described m Example 3 are evaluated by the method desaibed below. Nine hundred microliter (900 [iL) of the yeast suspenaons cultured by the method described in [Bcample 4 and prepared as OD660=2 are di^ensed into two microtubes, re^ectively. One hundred microHter (100 pL) of staile water is added to OIK of the microtubes, on the other hand, 100 pL of 99.5 % ethanol is added to another one (final concenti^on is 10%). The su^ensions are stored at 5°C for 4 weeks, then viable cell ratios are measured by the same method as Example 4.
INDUSTRIAL APFLICABILirY

According to the present invention, yeast can be stored stably for emended period of tim_, because drying-resistant propaty and/or low-temperature storage-resistant property can be enhanced by the present invention. AccordJi^y, efficiency of brewing alcoholic bever^es (such as beer), production of bread, or manufecturir^ usefiil materials such as industrial alcohol production OT production of usefial prolans, etc., can be in^sroved by Ihe i^esent invention.

CLAIMS
1. A polynucleotkie selected from the group
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ1DN0:1;
(b) a polynucleolide comprising a polynucleotide encoding a protein of the amino add sequence of SEQ E3 NO; 2;
(c) a polynucleotide conqirising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in white one or more amino adds thereof are deleted, substituted, inserted and/or added, and having dephosphoiylaiion activity;
(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence 60yo or higher identity with the amino acid sequence of SEQ ID NO: 2, and said protein having a trehaiose-6-phosphate dephosphoryktion activity;
(e) a polynucleotide comprising a polynucleotide which hybridize to a polynucleotide of a nucleotide sequence complementary to the nude tide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein having a trehalose-6-phosphate dephosphorylation activity, and
(f) a polynucleotide comprising a polynucleotide via hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to thb nudeotide sequence of the polynucleotide encoding the protein havu^ the amino add sequence of SEQ ID NO: 2 under stringent conditions, and which encodes a protein having a ti:ehalose-6~phosphate dq)hospbo]ylation activity.
2, The polynucleotide according to Claim 1 sdected from the group consisting of
(g) a polynucleotide conq^rising a polynucleotide Kicodii^ a protein consisting of the
amino acid secpience of SEQ ID NO: 2, or aicoding the amino add sequMice of SEQ ID NO: 2 in
which 1 to 10 amino adds the-eof are deleted, substituted, mserted, and/or added, and wherein said
protein has a trehalose-6-phosphate dephosphorylation activity;
(h) a poiynudeotide coroprisii^ a polynudeotide aico(fing a protein ha^ong 90% or Mgher identity with the amino add sequoice of SEQ ID NO; 2, and having a trehalose-6-phosphate dephosphoryktion activity, and
(i) a poiynudeotide comprising a polynucleotide w^iich hybridizes to a polynucleotide consisting of a nudeotide sequence of SEQ ID NO; 1 or which hybridizes to a polynucleotide consisting of a nudeotide sequence complementary to the nudeotide sequence of SEQ ID NO: 1, unda' hi^ stringait conditions, which encodes a protein having a trehalose-6-phog)hate dephosphorylation activity.

3. The polynucleotide according to Claim 1 comprising a polynucleotide consistir^ of the nucleotide sequence of SEQ E) NO: 1.
4. The polynucleotide according to Claim 1 oan^rising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ 3D NO: 2.
5. The polynucleotide according to any one of Claims 1 to 4, vdiereiu the polynucleotide isDNA.
6. A prolan encoded by the polynucleotide acccffdii^ to ai^ one of Claims 1 to 5.
7. A vector containing the polynucleotide ^xording to any one of Claims 1 to 5.
8. A yeast into which the vector accordmg to Claim 7 has been introduced.
9. The yeast accordii^ to Claim 8, wherao diying-resistant prt^erty is increased.

10. Theyeast according to Claims, wiierean Ibw-temperature storage-redstant property is inaeased
11. The yeast according to Claim 9, wdiadn the drying-reastant propaty is increased by increasir^ an Kq)ression level (rfthe protean of Claim 6.
12. The yeast acccffding K> Claim 10, wheran flie low-tanperature storage^esistant propaty is increased by increasing an expression level of the prolan of Claim 6.
13. A method for i^oducu^ an alcohohc beverage by usuig the yeast according to any one of Claims 8to 12.
14. The mairod according to Chiim 13, ifiiiam liie brewed alcoholic beverage is a malt beverage.
15. The method according to Claim 13, vAra-ein the brewed alcoholic beverage is wine.

16. An alcoholic beverage produced by the method according to ar^' one of Claims 13 to
15.
17. A method fcr assessing a test yeast for its diying-resistant jM-operty and/or
low-temperature storage-resistant property, comprising uang a primer or probe designed based on
the nucleotide sequence ofa gene having the nucleotide sequKiceofSEQ ID NO: 1 and encoding a
trehalose~6-phosphate phosphatase.
18. A method for assessing a test yeast for its drying-resistant property and/or
low-temperature storage-reastant properly, comprising: culturing the test yeast; and measuring the
e^resaon level of the gaie having the nucleotide sequence of SEQ ID NO: 1 and encoding a
trehalDse-6-phosphate phosphatase.
19. A method for selecting a yeast, conprising: ailturing test yeasts; quantifying the
protein of Clahn 6 or measuring the ejqiression level of the gene havii^ the nucleotide sec^ience of
SEQ ID NO: 1 and encoding a trehalose-6-phosphate phosphatase; and selecting a teS yeast having
an amount of the protdn or the gaie expression level acconJing to fevorable drying-resistant
propa^ and/or low-temperature storage-resistaitt property.
20. The method for selecting a yeast according to Claim 19, con^rising: culturing a
refe'fflice y^st and test yeasts; measurii^ for each yeast the e^ession level of the gene having the
nucleotide sequence of SEQ ID NO: 1 and encoding a trdialose-6^hosphate phosphatase; and
selecting a test yeast having the gene expression h^her than that in the reference yeast.
21. The method for selecting a yeast accordii^ to Claim 19, comprising; culturing a
reference yeast ai^ test yeasis; quantifyii^ the prolan accordii^ to Claim 6 in each yeast; and
sdecting a test yeast having a larger amount of the protein than that in the reference yeast.
22. A method iir producii^ an alcohohc beverage comprising: conducting fermentation
using tiie yeast according to any one of Cldms 8 to 12 or a ye^ selected by the methods according
to any one of Claims 19 to 21.